Effects of mutagenesis of residue 221 on the properties of bacterial and mitochondrial elongation factor EF-Tu $ Senyene Eyo Hunter, Linda L. Spremulli * Department of Chemistry, Lineberger Cancer Research Center, University of North Carolina, Campus Box 3290, Chapel Hill, NC 27599-3290, USA Received 8 September 2003; received in revised form 14 January 2004; accepted 19 February 2004 Available online 28 March 2004 Abstract During protein biosynthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of ribosomes. This factor is highly conserved throughout evolution. However, several key residues differ between bacterial and mammalian mitochondrial EF-Tu (EF- Tu mt ). One such residue is Ser221 (Escherichia coli numbering). This residue is conserved as a Ser or Thr in the bacterial factors but is present as Pro269 in EF-Tu mt . Pro269 reorients the loop containing this residue and shifts the adjoining h-strand in EF-Tu mt compared to that of E. coli EF-Tu potentially altering the binding pocket for the acceptor stem of the aa-tRNA. Pro269 was mutated to a serine residue (P269S) in EF-Tu mt . For comparison, the complementary mutation was created at Ser221 in E. coli EF-Tu (S221P). The E. coli EF-Tu S221P variant is poorly expressed in E. coli and the majority of the molecules fail to fold into an active conformation. In contrast, EF-Tu mt P269S is expressed to a high level in E. coli. When corrected for the percentage of active molecules, both variants function as effectively as their respective wild-type factors in ternary complex formation using E. coli Phe-tRNA Phe and Cys-tRNA Cys . They are also active in A-site binding and in vitro translation assays with E. coli Phe-tRNA Phe . In addition, both variants are as active as their respective wild-type factors in ternary complex formation, A-site binding and in vitro translation assays using mitochondrial Phe-tRNA Phe . D 2004 Elsevier B.V. All rights reserved. Keywords: Mitochondria; Bacteria; Protein synthesis; Elongation; Elongation factor Tu; Mutagenesis 1. Introduction During protein biosynthesis, elongation factor Tu (EF-Tu), in its active GTP conformation, binds aminoacyl-tRNA (aa- tRNA) and promotes its binding to the A-site of the ribosome [1]. Upon positive codon/anticodon interactions, GTP is hydrolyzed to GDP and the inactive EF-TuGDP complex dissociates from the ribosome, leaving the aa-tRNA in the A- site. After dissociation, the guanine nucleotide exchange factor, elongation factor Ts (EF-Ts), displaces GDP and binds EF-Tu. EF-Ts is then displaced by GTP, thus reactivating EF- Tu and allowing the formation of another ternary complex. Mammalian mitochondria have a translational system with both similarities and differences to the prokaryotic system. Mitochondrial EF-Tu (EF-Tu mt ) functions in translation in a manner analogous to the bacterial factors. However, the binding constants that govern the interactions between EF- Tu and its ligands differ significantly between Escherichia coli EF-Tu and EF-Tu mt [2]. The only structure of EF-Tu mt currently available is that of bovine EF-Tu mt in complex with GDP which shows that the structure of the mitochondrial factor is very similar to that of its prokaryotic counterparts [3]. One of the most unusual features of the mammalian mitochondrial translational system is its tRNAs. The prima- ry sequences of most tRNAs contain conserved or semi- conserved residues that enable the tRNAs to fold into stable ‘‘L’’shaped structures. Canonical tRNAs also contain a high G–C content in their stem regions to improve their stabil- ities [4]. Mitochondrial tRNAs lack a number of the invariant and semi-invariant residues found in other tRNAs. Furthermore, they have a high A–U content in their stem regions and are often significantly shorter than canonical tRNAs, having as few as 59 nucleotides compared to 75 or more in other tRNAs [3,4]. No direct structural information is available on these tRNAs, although chemical probing and NMR analysis suggest that they do form a basic L-shape [5]. 1570-9639/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2004.02.015 $ This work has been supported in part by funds provided by the National Institutes of Health (Grant GM32734). * Corresponding author. Tel.: +1-919-966-1567; fax: +1-919-966- 3675. E-mail address: Linda _ Spremulli@unc.edu (L.L. Spremulli). www.bba-direct.com Biochimica et Biophysica Acta 1699 (2004) 173 – 182